CN1177375A - New strains of Exserohilum monoceras and their uses - Google Patents

Abstract

A herbicide and a herbicidal method with the use of a strain belonging to Exserohilum monoceras which shows an esterase zymogram pattern differing from those of the conventional microorganisms and has a high capability of forming spores.

Description

New strains of Exserohilum monoceras and their uses

The present invention relates to a novel strain of Exserohilum monoceras, a weed control agent containing the strain, and a weed control method using the weed control agent.

In order to stabilize food production, it is necessary to use pesticides in farmland. However, due to the long-term application of large quantities of synthetic pesticides containing non-naturally occurring substances to the land, these substances can have harmful effects. The most serious problem is that it directly affects people and livestock, and damages the ecosystem. In recent years, since the specificity of pesticides for weeds, harmful insects and pathogens has been greatly improved, their harmful effects on humans and domestic animals have been reduced. However, adequate attention should still be paid to these adverse effects in relation to the high pesticide concentrations when pesticides are used. Moreover, the selection of the concentration of pesticides for weeds, harmful insects and pathogens need not be high, and high concentrations often lead to damage to the ecosystem.

To solve this problem, some herbicides need to be developed to protect the environment. These herbicides do not adversely affect species other than the target species. An example of the above-mentioned herbicide is a herbicide using a weed pathogen. Certain weed pathogens are ubiquitous in weeds, and it is known that these pathogens can be used in herbicides which hardly harm humans, domestic animals, and small organisms including fish, insects, etc. Plants are less phytotoxic. Furthermore, the specificity of this herbicide for the use of pathogens on target weeds is so high that its selectivity is quite high, with little damage to the ecosystem. Currently, commercially available examples of herbicides using weed pathogens are the herbicide Devine using Phytophthora palmivora for Stranglervine, the variant aeschynomene of Colletotrichum gloeosporioides The herbicide Collego for Northern joint vetch and the herbicide BioMal for Malvae roundleaf using Trichospora disciflorum var. malvae.

An example of a herbicide using a weed pathogen to control barnyardgrass (Echinochloa spp.), the most serious weed in rice fields, is Cochliobolus lunatus (Anamorph: Curvularia lunata) [Weed Research, 27, 43-47 , (1987) and Japanese Patent Application Publication No. 284,963/93], Ustilago trichophora [WO93/05656] and Drechslera monoceras (synonym of Exserohilum monoceras) [Japanese Patent Application Publication Series 219,883/91, 226,905/92, 360,678/92, 370,090/92, 277,042/94, 329,513/94 and 247,822/94].

But these conventional herbicides have limitations: for example, Cochliobolus lunatus needs a wetting period of more than 18 hours to fully show its effect; Ustilago trichophora needs 4 to 5 weeks after spraying to fully show its effect; and Drechslera monoceras produces A small number of spores. Therefore, herbicides using these fungi have not yet come into practical use.

It is an object of the present invention to provide microorganisms having both sufficient herbicidal action and high sporulation performance.

The inventors screened strains pathogenic to barnyard barnyardgrass, obtained a strain with strong herbicidal effect, and found a strain with both strong herbicidal effect and high spore-producing performance. In addition, the inventors detected the esterase zymogram types of these strains and found that such strains exhibited similar esterase zymogram types, which were not similar to all esterase zymogram types of known strains. Based on these findings, the present inventors have accomplished the following inventions:

The present invention is a strain of Exserohilum monoceras, which exhibits the type of esterase zymogram shown in Figure 1.

In addition, the present invention is a weed control agent containing said strain as an active ingredient.

Furthermore, the present invention is a method of controlling weeds comprising using the weed control agent.

Hereinafter, the present invention is described in detail.

The bacterial strain of the present invention is isolated from diseased barnyardgrass, and the characteristics of the fungus are as follows:

It is an aerobic organism that forms dark gray or black colonies on potato sucrose agar plates. White or gray aerial mycelium can be observed. The fungus has a large number of dark-colored conidia, each spore has 2-8 septa, is spindle-shaped, widest in the middle, and narrows toward the end. A door protrudes from the base end. The size of conidia is about 40-150×10-25 μm.

From the above (observation) results, especially the formation and shape of its bacterial colony and the morphology of its conidia, by referring to A. Sivanesan: "The herbaceous species of the genus Bipolaris, Curvularia, Drechslera, Exserohilum and their variants" (Graminicolous Species of Bipolaris, Curvularia, Drechslera, Exserohilum and Their Telemorphs) ("Mycological Papers", No.158, P.261, Nov.1987) pp. 201-237, the inventor will The strain was identified as Exserohilum monoceras.

The name "Exserohilum" for this fungal genus is according to the classification method of A, Sivanesan. This classification is also supported by Luttrell (Revue de Mycologie, 41, 271-279, (1977)) and Alcom (Mycotaxon, 8, 411-414, (1978). In On the other hand, Ellis (Dematioceous Hyphomycetes, CMI, Kew, 608 (1971)) once classified the genus Exserohilum and Bipolaris into the genus Drechslera, and only used the genus Drechslera. However, in recent years, no researcher except Ellis has not It is agreed that there are Exserohilum and Bipolaris species, therefore, it is considered suitable to use Exserohilum species. Since the deformation state of Exserohilum species is known as Setosphaeria species, microorganisms classified as Setosphaeria species can also be included within the scope of the present invention.

The strains of the present invention are specifically JTB-012, JTB-013, JTB-799, JTB-803 and JTB-808. JTB-012 has been deposited as FERM BP-5271, JTB-013 as FERM BP-5272, JTB-799 as FERM BP-5273, JTB-803 as FERM BP-5274, JTB-808 as FERM BP-5275, all strains Deposited on October 27, 1995 at the Agency for Industrial Science and Technology, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi l-chome, Tsukuba-shi , Ibaraki-Ken, Japan).

As shown in Figure 2, these strains exhibited an esterase zymogram pattern that was different from that of the known strains IFO9800, IMI125854, IMI125855, ATCC24641 and ATCC58346 of Exserohilum monoceras.

The strain of the present invention can be cultivated in the same manner as for known strains of Exserohilum monoceras without special methods. The medium may be any synthetic or natural medium so far as it suitably contains assimilable carbon and nitrogen sources and inorganic materials and necessary growth promoting agents. Examples of the medium are oat sucrose agar medium, oat agar medium, potato sucrose medium, V-8 juice agar medium, Czapek-Dox agar medium and the like. During the cultivation, the medium is kept at a temperature of 15-30°C, preferably at 20-25°C, and the medium is kept at pH 3-9, preferably at pH 5-8. After 7-14 days of culture under the above conditions, a sufficient number of spores are formed on the medium plate.

The weed control agent of the present invention is prepared by adding a surfactant or the like to the spores as an active ingredient. The spore density can be determined arbitrarily within the range where herbicidal effect can be exhibited, and within this range, a density of 10 ² -10 ⁶ spores/ml, preferably 10 ³ -10 ⁵ spores/ml can be suitably used.

For using the weed control agent of the present invention in an actual field, it is preferable to use a density of 10 ⁹ -10 ¹⁰ spores/1000 m ² .

Weeds to be treated with the weed control agent of the present invention include barnyardgrass, but are not limited thereto.

Example [Example 1] Control experiment of barnyardgrass (Echinochloa crus-galli)

Infected plants of Echinochloa spp. were collected from various places in Japan. The infected plaques were excised and cultured under humid conditions at 25°C, thereby forming conidia. Conidia were scraped off with a needle and inoculated on a potato sucrose agar medium plate to isolate individual spores. All strains were inoculated on oat sucrose agar medium plates and cultured at 25°C for 14 days to form conidia. Then, conidia were suspended in a 0.02% aqueous solution of Tween 20 at a density of 10 ³ , 10 ⁴ and 10 ⁵ spores per 5 ml.

Cultivate Barnyardgrass barnyardgrass to the 1.5-leaf stage in a 100cm ² pot, and fill with water to a depth of about 5cm. 5 ml of each of the spore suspensions was added drop by drop to each pot, and kept in the greenhouse for 3 weeks, and the herbicidal efficiency was detected. The herbicidal efficiency against barnyardgrass was determined as follows:

Herbicide efficiency=[1-(number of surviving individuals/20)]×100

Table 1. Strains and their herbicidal efficiency against barnyard barnyardgrass

10 ³ 10 ⁴ 10 ⁵ JTB-012 38 95 100JTB-013 35 70 95JTB-799 47 100 100JTB-803 35 100 100JTB-808 32 100 100IMI-125855 0 2 25 Untreated 0 [Example 2] Propagation of conidia force experiment

Each strain was inoculated on an oat sucrose agar medium plate, and cultured statically at 25°C for 14 days. The conidia on the medium plate were then recovered by suspending them in 0.1% Tween 20. The amount of conidia produced is shown in Table 2.

Table 2. Strains and their conidia production

Strain Conidia production/cm ³

JTB-012 4.4×10 ⁵

JTB-013 4.5×10 ⁵

JTB-799 7.5×10 ⁵

JTB-803 8.6×10 ⁵

JTB-808 6.6×10 ⁵

IFO-9800 <2.4×10 ³

IMI-125854 <2.4×10 ³

IMI-125855 4.0×10 ⁴

ATCC-24641 <2.4×10 ³

ATCC-58346 <2.4×10 ³

Strains such as JTB-012, JTB-013, JTB-799, JTB-803 and JTB-808 produced conidia at least at a yield of 4.4×10 ⁵ conidia/cm ³ . On the other hand, IMI-125855, the strain most capable of producing conidia among known strains, produced conidia at a yield of 4.4×10 ⁴ conidia/cm ² . The amount of conidia produced by all other known strains was below the detection limit, indicating that the amount of conidia produced by each of these strains was 10 times higher than that of each known strain. [Example 3] Esterase zymogram analysis

Ten strains of JTB-012, JTB-013, JTB-799, JTB-803, JTB-808, IFO-9800, IMI-125854, IMI-125855, ATCC-24641 and ATCC-58346 were used as samples. A crude enzyme solution was prepared from each strain by the method described in Japanese Patent Application Laid-Open No. 329,513/94.

Each strain was cultured statically in potato sucrose broth at 25°C in the dark for 7-10 days to form fungal clusters. The fungal mats were washed several times with distilled water, frozen at -80°C, and lyophilized.

The fungal flora was homogenized in 50 mM Tris-HCl buffer (pH 7.4), filtered through filter paper, and the filtrate was centrifuged at 10,000 r.p.m. The supernatant was used as a sample. The concentration of sample protein was quantitatively detected by the Lowry method. Each sample (about 50 μg protein) was electrophoresed at 30 mA for 2 hours using an acrylamide gel (stacking gel, 4.5%; separating gel, 10%) in a large slab gel electrophoresis tank.

After electrophoresis, esterase activity staining was performed. 40 mg of naphthyl acetate was dissolved in an aqueous solution of 50% acetone, 200 mg of fast violet B salt and 200 ml of 50 mM Tris-HCl buffer were added thereto, and the resulting solution was used as a staining solution. Submerge the gel in the staining solution and shake slightly for 30 minutes for staining. Then, the gel was washed with distilled water and the mobility in each band was examined.

As a result, as shown in Figures 1 and 2, JTB-012, JTB-013, JTB-799, JTB-803 and JTB-808 showed similar esterase zymogram types, therefore, it is clear that JTB-012, JTB-013 , JTB-799, JTB-803 and JTB-808 strains belong to the same class. Due to the esterase zymogram type of such strains and the known IFO-9800, IMI-125854, IMI-125855, ATCC-24641, ATCC-58346 and Drechsrela monoceras var described in Japanese Patent Application Publication No. 329,513/94 .. microsporus strains, it is also clear that these strains are a new class of strains different from the known strains. [Examples of preparations] Example 1 (emulsion formed in water)

2×10 ⁹ conidia of Exserohilum monoceras and 4 g of Tween 80 were added to 20 L of sterile water and mixed to prepare a liquid preparation. Example 2 (wettable powder)

Suspend conidia (JTB-803) in a mixture of 9% maltose, 1% clay and 90% water to prepare a suspension containing 10 ⁷ conidia per ml. The suspension is air-dried, and the dried product is ground to make a wettable powder. Example 3 (wettable powder)

Suspend conidia (JTB-012) in a mixture of 9% lactose, 1% zeolite and 90% water to prepare a suspension containing 10 ⁷ conidia per ml. The suspension is air-dried, and the dried product is ground to make a wettable powder. Example 4 (wettable powder)

Suspend conidia (JTB-808) in a mixture of 15% diatomaceous earth, 77% kaolin and 8% polyvinyl alkylphenyl ether to make a suspension containing ¹⁰⁷ conidia per ml . The suspension is air-dried, and the dried product is ground to make a wettable powder. Example 5 (wettable powder)

Suspend conidia (JTB-799) in a mixture of 33% diatomaceous earth, 0.33% carboxymethylcellulose and 66.67% water to prepare a suspension containing 10 ⁷ conidia per ml. The suspension is dried, and the dried product is ground to prepare a wettable powder. Example 6 (powder)

The conidia (JTB-012) were mixed with a mixture containing 14% hydroxypropyl-β-cyclodextrin, 12% white carbon black and 74% clay to make ¹⁰⁷ fractions per g. The spore-forming mixture is dried, ground evenly, and made into a powder. Example 7 (granules)

Knead conidia (JTB-808) with a mixture containing 15% β-cyclodextrin, 2% starch, 18% bentonite, 36% potassium carbonate and 29% water to make The mixture containing ¹⁰⁷ conidia is then granulated in a granulator and dried to make granules. Example 8 (emulsifiable concentrate)

Evenly suspend the conidia (JTB-799) in a mixture containing 18% polyoxyethylene nonylphenyl ether ammonium phosphate, 6% polyoxyethylene nonylphenyl ether, 29% triphosphate Ethyl ester and 47% tributyl phosphate make an emulsion containing 10 ⁷ spores per ml. Example 9 (oil agent)

Conidia (JTB-803) were suspended in a mixture containing 95% spindle oil, 4% castor oil and 1% silicone oil to make an oil preparation containing ¹⁰⁷ spores per ml. Example 10 (dry flowable)

Suspend conidia (JTB-013) in a composition containing 12% sodium alkylbenzene sulfonate and 88% polyethylene glycol ether to make ¹⁰⁷ meristems per ml Dry mobile agent of spores. Example 11 (microcapsules)

The conidia (JTB-803) were suspended in a mixture containing 0.7% 8-sodium alginate, 5% kaolin, 15% glycerol and 79.3% water to make ¹⁰⁷ conidia per ml Suspension. The suspension was added dropwise onto 0.2M calcium acetate to produce microencapsulated products. The product is chopped, sieved and air-dried to make microcapsules. Example 12 (microcapsules)

Suspend conidia (JTB-013) in a mixture containing 0.7% sodium alginate, 5% kaolin, 15% glycerol and 79.3% water to make a suspension containing ¹⁰⁷ conidia per ml. liquid. The suspension was added dropwise onto 0.2M calcium chloride to produce microencapsulated products. The product is chopped, sieved and air-dried to make microcapsules.

The present invention provides a new strain of Exserohilum monoceras. Compared with the conventional strain of Exserohilummonoceras, the strain has good herbicidal effect and spore reproduction ability, and has suitable performance as a component of fungal herbicide.

Brief description of the attached drawings:

Fig. 1 is the esterase zymogram of new bacterial strain of the present invention

Fig. 2 is the esterase zymogram of the new bacterial strain of the present invention and conventional bacterial strain.

Claims (6)

1.Exserohilum a kind of bacterial strain of monoceras, this strains expressed have the esterase zymogram shown in Fig. 1.

2. bacterial strain as claimed in claim 1, this bacterial strain are Exserohilum monoceras JTB-012, Exserohilum monoceras JTB-013, Exserohilum monoceras JTB-799, Exserohilum monoceras JTB-803 or Exserohilum monoceras JTB-808.

3. preparation that is used to control weeds, said preparation includes as the claim 1 of active ingredient or 2 bacterial strain.

4. preparation as claimed in claim 3, wherein these weeds are barnyard grass (Echinochloa spp).

5. one kind is used to control method for weed, and this method comprises the preparation that uses claim 3.

6. method as claimed in claim 5, wherein these weeds are barnyard grass (Echinochloa spp).

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